Journal: PLOS Genetics

Article Title: METTL3 facilitates the translation of CircSIK2 during chicken myogenesis in an m 6 A dependent manner

doi: 10.1371/journal.pgen.1011934

Figure Lengend Snippet: ( A) Details of seven potential methylation modification sites on circSIK2. (B ) The relative expression of circSIK2, METTL3, and SIK2 mRNA in myoblast cells after transfected with pCD25-circSIK2 + pCD3.1, pCD25-circSIK2 + pCD3.1-METTL3, and pCD25 + pCD3.1. ( C) Upper: the expression of SIK2-176aa-flag after transfected with pCD3.1-SIK2-176aa-flag and pCD3.1-SIK2-176aa-flag + pCD3.1-METTL3. Lower: the expression of SIK2-176aa after transfected with pCD3.1-SIK2-176aa-flag, pCD25-circSIK2, and pCD25-circSIK2 + pCD3.1-METTL3. (D ) Dual-luciferase reporter assay using the different regions of the circSIK2 after co-transfected with METTL3 in DF-1 cells. ( E) Upper: Dual-luciferase reporter assay using the 151–200 fragment after co-transfected with METTL3 in DF-1 cells. Under: Dual-luciferase reporter assay after 131A-WT and 131A-MT fragment was co-transfected with METTL14 in DF-1 cells. (F ) Amplification curve and qPCR CT value in circSIK2 131 m 6 A site and 125 A site after METTL3 overexpression in myoblast cells. ( G, H) circSIK2 pulldown image ( G ) (OV = pCD25-circSIK2, NC = pCD25) and binding proteins’ string network ( H ) screened by mass spectrometry in myoblast cells. ( I ) circSIK2 pulldown results confirmed by western blot with HNRNPA2B1 antibody in myoblast cells. (J) The expression pattern of METTL3, METTL14, EIF3A, and EIF3F in different stages of myoblast differentiation. GM (growth media; n = 3) stands for myoblasts in the proliferative phase. DM (differentiation media; n = 3), DM1-DM6 means differentiation from day 1 to day 6.

Article Snippet: The full length of the METTL3 coding sequence (NCBI Reference Sequence: XM_040655036.2 ) was also synthesized by Sangon Biotech and cloned into the pcDNA3.1 (+) vector.

Techniques: Methylation, Modification, Expressing, Transfection, Luciferase, Reporter Assay, Amplification, Over Expression, Binding Assay, Mass Spectrometry, Western Blot

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Optimization of pGC-A expression in Sf9 cells. Western blot (anti-pGC-A) of total cell protein over different virus multiplicities of infection (MOI) and transfection time. MOI of 0 indicates no virus transfection. pGC-A (red arrow) is approximately 120 kDa.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Determination of expressed full-length pGC-A functionality via whole-cell activity assay. The competitive ELISA assay measured the cGMP yield level in Sf9 cells expressing full-length pGC-A versus control Sf9 cells (n = 2). Both cell types were incubated with different concentrations of MANP ligand (0 to 10 7 pmol). Incubation with 0 pmol MANP served as a negative control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Activity Assay, Competitive ELISA, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purification of full-length pGC-A via affinity and size exclusion columns. ( A ) Western blot (anti-pGC-A) of samples from cell lysis to the affinity column. pGC-A (red arrow) is ~ 120 kDa. ( B ) Coomassie blue stain from cell lysis to the affinity column. ( C ) Coomassie blue stain of the eluted fraction from Superose 6 that was used for protein crystallization. M: marker; P: membrane pellet from ultracentrifugation after solubilization with n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS); FT: flowthrough from the affinity column; 50, 100, 500: imidazole (mM) at elution from the affinity column.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, Western Blot, Lysis, Affinity Column, Staining, Crystallization Assay, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Presence of pGC-A in crystals was confirmed via western blot. The anti-pGC-A antibody was used in the western blot. The pGC-A monomer is 120 kDa. Lane 1: combined mix: combined crystallization drops collected in the PCR tube. Lane 2: supernatant: the supernatant collected from the centrifuged combined mix. Lane 3: washed pellet: the crystal pellet was washed with the precipitant solution and collected again via centrifugation. Lane 4: washed supernatant: the supernatant from washed crystal pellet. Lane 5: crystallization drop: one crystallization hanging drop directly mixed with SDS sample buffer. Lane 6: crystallization sample: purified pGC-A before crystallization, serving as a positive control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Western Blot, Crystallization Assay, Centrifugation, Purification, Positive Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Indexed diffraction patterns from pGC-A microcrystals collected via serial crystallography at the Advanced Photon Source. Three indexable diffraction patterns with the highest resolution of 3 Å. ( A ) Blue/pink diffraction graphs show diffraction patterns analyzed using CrystFEL software. ( B ) The original diffraction patterns shown with white background. All diffraction dots were manually circled in red for better visualization.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Software

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Dynamic oligomeric states of pGC-A seen in replicate runs of Superose 6 size exclusion chromatography may be dependent on protein concentration. ( A ) The Superose 6 10/300GL column performance profile. Five standard proteins were used to generate relative molecule elution points based on different molecular sizes. Thyroglobulin (669 kDa) eluted at 14.19 mL, ferritin (440 kDa) eluted at 15.96 mL, aldolase (158 kDa) eluted at 17.58 mL, ovalbumin (44 kDa) eluted at 18.49 mL, and aprotinin (6.5 kDa) eluted at 21.60 mL. ( B-D ) Size exclusion chromatography of pGC-A. ( B ) The peak intensity at 17.3 mL corresponds to the pGC-A monomeric state (120 kDa). pGC-A monomer is the major peak determined by chromatography. Other ratios were faded out in the background and served as supplemental comparison. ( C ) pGC-A tetramer and monomer present similar ratios in the chromatographic separation. The peak intensity at 14.76 mL and 17.29 mL corresponds to pGC-A tetrameric (480 kDa) and monomeric states, respectively. ( D ) The pGC-A tetramer is the major peak. The peak intensity at 15.05 mL corresponds to the pGC-A tetrameric state.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Size-exclusion Chromatography, Protein Concentration, Chromatography

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stain of high resolution clear-native PAGE from pGC-A Superose 6 fractions. Superose 6 column eluted tetramer (480 kDa) and monomer (120 kDa) size peaks were concentrated and analyzed in a 4–16% native gel. M, marker.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Silver Staining, Clear Native PAGE, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stained clear-native PAGE of different oligomeric samples treated with or without dithiothreitol (DTT) overnight. Three peak samples, which represent the tetramer, dimer, and monomer of full-length pGC-A, were concentrated and split in half for treatment with or without DTT. S, concentrated sample only; S + DTT, concentrated sample incubated with 1 M DTT overnight.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Staining, Clear Native PAGE, Incubation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purified full-length pGC-A in vitro functional activity test. ( A ) Functional activity for two pGC-A oligomeric states with ATP incubation. The control group was GTP and ATP in sample buffer. For activity values in units of mg of purified pGC-A, the y-axis values of pmol/mL can be converted to nmol/mg protein by multiplying by 0.00873. ( B ) Functional activity for two pGC-A oligomeric states without ATP incubation. The control group was GTP only in the sample buffer. ( C ) Competitive cGMP ELISA standard fit in four parameters logistic (4PL) curve. The left Y-axis is the B/B0 (%) value and represents the percentage of bound cGMP. The right Y-axis represents the average net optical density (OD) reading at 405 nm. Both standard curves were generated with a 95% confidence interval. ( D ) The cGMP yield differences between pGC-A oligomer samples incubated with or without ATP were analyzed. All raw data points were analyzed via the ROUT method (Q = 1%) to remove significantly impossible outlier values before data analysis. One-way ANOVA was used to determine the statistical significance between samples and control in graphs ( A ) and ( B ). Two-way ANOVA was used to determine the statistical significance in graph ( D ). Each dot represented to the sample point and plotted as mean ± standard deviation (SD). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 and ns P ≥ 0.05, Not significant.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, In Vitro, Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Proposed native state and three-step mechanism of full-length pGC-A. First, the full-length pGC-A forms a tetramer complex in the native state by non-covalent interactions (e.g., hydrogen bond and hydrophobic interactions). In each tetramer complex, there are two functional units, and each functional unit may represent a dimer. The narrowest part of the tetramer is the transmembrane domain. Second, the pGC-A signal transduction mechanism is not ATP-dependent. The current ATP-dependent two-step activation mechanism should instead be three-step. The first step is ligand (ANP) binding, which moderately activates the pGC-A; the second step is binding ATP, which partially boosts protein activity; the third step is the pGC-A phosphorylation, which fully activates the guanylyl cyclase.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Functional Assay, Transduction, Activation Assay, Binding Assay, Activity Assay

Journal: Nature

Article Title: Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

doi: 10.1038/nature13441

Figure Lengend Snippet: Figure 2 | Mutant IDH blocks hepatocyte differentiation by silencing HNF- 4a. a, Heat map of GSEA showing top-ranked gene sets distinguishing IDH1(R132C) or IDH2(R172K) from wild-type (WT) or empty vector (EV) control hepatoblasts (pairwise analysis; replicates for each condition; see Methods). NES, normalized enrichment score; NS, not significant. b, c, Hepatoblasts analysed by immunoblot (b) and qRT–PCR (c). d, e, Analysis of wild-type hepatoblasts expressing the indicated short hairpin (sh)RNAs (uncoated plates). CTL, control. d, Hepatocyte sphere formation. e, Proliferation of shRNA-expressing hepatoblast cells co-expressing EV or shRNA-resistant Hnf4a(1) complementary DNA. f–h, Control and R132C- expressing hepatoblasts co-expressing vector control (EV2) or HNF-4a, grown on uncoated plates. f, Hepatocyte sphere formation. g, Hepatocyte gene expression. h, Proliferation. Scale bars, 100mm (d), 250mm (f). *P , 0.05.

Article Snippet: The R140Q and R172K mutations were introduced into human IDH2 cDNA (Origene, MD # SC319226, NCBI RefSeq accession number NM_002168.3) using the Quickchange kit (Agilent Lexington, MA #200521) as per the manufacturer’s instructions.

Techniques: Mutagenesis, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Expressing, shRNA, Gene Expression

Journal: Molecular medicine reports

Article Title: IRX2-mediated upregulation of MMP-9 and VEGF in a PI3K/AKT-dependent manner.

doi: 10.3892/mmr.2015.3915

Figure Lengend Snippet: Figure 1. Increased expression of IRX2 was observed in osteosarcoma tissue samples, determined using western blot analysis. (A) Results of western blot analysis for the expression of IRX2 in eight paired osteosarcoma and corresponding normal tissue samples. (B) Proteins were extracted from primary and metastatic tissue samples. β‑actin was used as a loading control. N, normal; P, paired osteosarcoma.

Article Snippet: The coding sequence of human IRX2 (NM_033267.4; NCBI Reference Sequence, OriGene Technologies, Rockville, MD, USA) was amplified by performing polymerase chain reaction (PCR) using the forward pr imer, 5'-GGATCCATGTCCTACCCGCAGGGC-3', which introduced a BamHI site, and the reverse primer, 5'-GAATTCCTATAGGTAGGGCTGGACGC-3' (Sangon Biotech Co., Ltd., Shanghai, China), which introduced an EcoRI site.

Techniques: Expressing, Western Blot, Control

Journal: Molecular medicine reports

Article Title: IRX2-mediated upregulation of MMP-9 and VEGF in a PI3K/AKT-dependent manner.

doi: 10.3892/mmr.2015.3915

Figure Lengend Snippet: Figure 3. IRX2 induces the phosphorylation of Akt and increases the expression levels of MMP‑9 and VEGF in osteosarcoma cells. Western blot analysis of the protein expression of VEGF, MMP‑9, p‑AKT and AKT in the indicated cells. β‑actin was used as an internal control. p‑, phosphorylated.

Article Snippet: The coding sequence of human IRX2 (NM_033267.4; NCBI Reference Sequence, OriGene Technologies, Rockville, MD, USA) was amplified by performing polymerase chain reaction (PCR) using the forward pr imer, 5'-GGATCCATGTCCTACCCGCAGGGC-3', which introduced a BamHI site, and the reverse primer, 5'-GAATTCCTATAGGTAGGGCTGGACGC-3' (Sangon Biotech Co., Ltd., Shanghai, China), which introduced an EcoRI site.

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: Molecular medicine reports

Article Title: IRX2-mediated upregulation of MMP-9 and VEGF in a PI3K/AKT-dependent manner.

doi: 10.3892/mmr.2015.3915

Figure Lengend Snippet: Figure 2. Increased expression of IRX2 promotes osteosarcoma cell growth and invasion in vitro. (A and B) Protein and mRNA expression levels of IRX2 were determined in MG63 and SaOS2 osteosarcoma cells transfected with IRX2 or a control vector. (C) An MTT assay was performed to assess the cell growth of MG63 and SaOS2 cells transfected with IRX2 or the control vector. (D) Representative images of the invasion assay of IRX2‑transfected osteosarcoma cells. The MG63 and SaOS2 cells were transfected with IRX2 and exhibited increased invasive activity, compared with the control vector‑transfected cells (magnification, x100). Quantitative analysis of cell invasion was assessed following 48 h of chemo‑attraction. Values represent the mean ± standard deviation of triplicates in one representative experiment. *P<0.05, compared with the control vector. OD, optical density.

Article Snippet: The coding sequence of human IRX2 (NM_033267.4; NCBI Reference Sequence, OriGene Technologies, Rockville, MD, USA) was amplified by performing polymerase chain reaction (PCR) using the forward pr imer, 5'-GGATCCATGTCCTACCCGCAGGGC-3', which introduced a BamHI site, and the reverse primer, 5'-GAATTCCTATAGGTAGGGCTGGACGC-3' (Sangon Biotech Co., Ltd., Shanghai, China), which introduced an EcoRI site.

Techniques: Expressing, In Vitro, Transfection, Control, Plasmid Preparation, MTT Assay, Invasion Assay, Activity Assay, Standard Deviation

Journal: Molecular medicine reports

Article Title: IRX2-mediated upregulation of MMP-9 and VEGF in a PI3K/AKT-dependent manner.

doi: 10.3892/mmr.2015.3915

Figure Lengend Snippet: Figure 4. PI3K/AKT are involved in IRX2‑induced cell proliferation, invasion and upregulation of MMP‑9 and VEGF. (A) Effects of Ly294002 on cell proliferation were measured using an MTT assay in MG63 and SaOS2 cells transfected with IRX2. (B) Effects of Ly294002 on cell invasion were assessed using an invasion assay in MG63 and SaOS2 cells transfected with IRX2. *P<0.05 and **P<0.01, vs. DMSO control. (C) MG63 and SaOS2 cells transfected with IRX2 were treated with or without Ly294002, and the expression levels of VEGF, MMP‑9, p‑Akt and AKT were detected using western blot analysis. p‑, phosphorylated. DMSO, dimethyl sulfoxide.

Article Snippet: The coding sequence of human IRX2 (NM_033267.4; NCBI Reference Sequence, OriGene Technologies, Rockville, MD, USA) was amplified by performing polymerase chain reaction (PCR) using the forward pr imer, 5'-GGATCCATGTCCTACCCGCAGGGC-3', which introduced a BamHI site, and the reverse primer, 5'-GAATTCCTATAGGTAGGGCTGGACGC-3' (Sangon Biotech Co., Ltd., Shanghai, China), which introduced an EcoRI site.

Techniques: MTT Assay, Transfection, Invasion Assay, Control, Expressing, Western Blot

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: The effect of time, temperature, and DNA/PEI ratio on mEGFP-MPP1 overexpression and HEK-293F cell viability and density. Flow cytometry analysis of the level of mEGFP-MPP1 overexpression after ( a ) 17 h transfection at 31 °C or ( b ) 5 h transfection at 37 °C using different DNA:PEI ratios. Error bars represent SD. Trypan blue-based analysis of the viability of cells transfected for ( c ) 17 h at 31 °C or ( d ) 5 h at 37 °C with different DNA:PEI ratios. The density of cells transfected for ( e ) 17 h at 31 °C or ( f ) 5 h at 37 °C with different DNA:PEI ratios. NT—non-transfected cells/ control; hpt—hours post-transfection. The procedure was performed in triplicate.

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Over Expression, Flow Cytometry, Transfection, Control

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: Expression and purification of MPP1 protein from HEK-293F cells. Western blot analysis of ( a ) crude cell lysates collected through transfection (0 h–72 h), samples collected after centrifugation (S1, P1) and purified recombinant protein (2 µg). The upper membrane was incubated with Anti-His-tag antibodies to detect overexpressed MPP1 and the lower membrane with Anti-GAPDH antibodies; ( b ) Coomassie blue staining of the SDS–PAGE gel with purified recombinant MPP1. The lower mass of purified recombinant MPP1 is a result of TAP-tag cleavage during the purification (for details, see ).

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Expressing, Purification, Western Blot, Transfection, Centrifugation, Recombinant, Membrane, Incubation, Staining, SDS Page

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: Characterization of the purified recombinant MPP1 protein using circular dichroism spectroscopy. ( a ) CD spectra of MPP1 protein 1, 3, and 7 days after dialysis recorded at 20 °C. ( b ) Thermal denaturation of MPP1 protein reflected via changes in ellipticity at 222 nm. The MPP1 was stored at 4 °C. Fu—fraction unfolded.

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Purification, Recombinant, Circular Dichroism, Spectroscopy

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: Identification of palmitoylation of overexpressed recombinant MPP1 protein by the Acyl-RAC method. Identification of palmitoylation of ( a ) overexpressed MPP1 protein in HEK-293F lysates and ( b ) recombinant MPP1 purified from HEK-293F cells. Both membranes were incubated with Anti-His-tag antibodies to detect recombinant MPP1. IF—input fraction (sample taken before the cleavage step); pF—preserved fraction; cF—cleaved fraction; PR—preservative reagent; CR—cleavage reagent.

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Recombinant, Purification, Incubation

Journal: The Journal of Experimental Medicine

Article Title: Identification of CD112R as a novel checkpoint for human T cells

doi: 10.1084/jem.20150785

Figure Lengend Snippet: Characterization of human CD112R protein. (A) Protein sequence encoded by the human CD112R gene. Predicted extracellular IgV-like and transmembrane domains are highlighted in blue and red, respectively. Two tyrosines (Y233 and Y293) in the cytoplasmic domain are underlined with one within an ITIM-like motif underlined. (B) Alignment of the extracellular domains of human and mouse CD112R protein sequences using the MacVector 6.5 program. The shaded boxes refer to the shared amino acids among CD112R orthologues. (C) Guide tree analysis of human CD112R and the known PVR-like proteins via the Clustal W program in MacVector 6.5. (D) Multiple sequence alignment of the IgV domains of PVR-like proteins. Similar and identical residues among this group are shaded in red. The PVR signature motifs are outlined in green frames. Blue boxes mark conserved amino acids. (E) A predicted protein structure model of human CD112R IgV domain (55–150 aa) using human PVRL4 (Protein Data Bank accession no. 4JJH ) as the template.

Article Snippet: Mouse CD112R cDNA (NCBI Reference Sequence accession no. XM_011240964 .1 ) was synthesized from GenScript and cloned onto a pcDNA3.1(−) expression vector.

Techniques: Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Identification of CD112R as a novel checkpoint for human T cells

doi: 10.1084/jem.20150785

Figure Lengend Snippet: CD112R expression in immune cells and its effect on TCR signal. (A) Human CD112R transcript in human immune cells. RNAs were isolated from DCs, NK cells, and T cells stimulated by OKT3 plus CD28 mAb. The expression of CD112R was detected by PCR. G3PDH was used as a housekeeping gene. (B) HEK293T cells transduced with control or CD112R gene were stained with control (red) or CD112R mAb (clone 2H6; blue). (C) Cell lysate of HEK293T/CD112R transfectant was run in reducing (+DTT) and nonreducing (−) conditions and detected by CD112R mAb (clone 2H6). (D) Flow cytometry analysis of CD112R expression in human peripheral blood from healthy donors ( n = 4 donors) stained with indicated cell surface markers. (E) CD112R expression on different NK cell subsets: CD16 + (CD56 + CD16 + ) and CD16 − (CD56 + CD16 − ). The expression of CD112R (blue) in these two NK subsets is shown. (F) The CD112R expression on CD4 + CD3 + and CD8 + CD3 + T cell subsets. Graph (right) shows mean ± SD frequencies of CD112R-expressing cells in each subset. (G) CD8 + T cells were divided into two groups based on the expression of CD112R, and their expression of CD45RA and CCR7 was revealed. (H) Purified CD4 + T cells were left unstimulated (day 0) or activated by anti-CD3/CD28 Dynabeads for different times, and the CD112R expression on T cells was detected by biotinylated CD112R mAb. (I) HEK293T cells were transiently transfected with WT or tyrosine mutants of CD112R. Cells were treated with or without pervanadate before analysis for tyrosine phosphorylation on CD112R. (J) Molt4 cell lysates were immunoprecipitated with CD112R mAb or mouse IgG1 (control) and blotted with different phosphatase mAbs as indicated. The presence of CD112R and tyrosine phosphorylation was demonstrated by immunoblotting with CD112R and phosphorylated tyrosine (P-Tyr) mAbs, respectively. Whole cell lysate serves as a detective control. (K) Jurkat-NFAT-Luc cells transfected with different chimeras as indicated were stimulated with OKT3 in the presence or absence of a mouse CD28 agonistic mAb. Data shows mean ± SD of relative luciferase activity upon 4 h of stimulation. All data shown are representative of at least two independent experiments. IP, immunoprecipitation. F and K were analyzed by Student’s t test; *, P < 0.05; **, P < 0.01.

Article Snippet: Mouse CD112R cDNA (NCBI Reference Sequence accession no. XM_011240964 .1 ) was synthesized from GenScript and cloned onto a pcDNA3.1(−) expression vector.

Techniques: Expressing, Isolation, Transduction, Control, Staining, Transfection, Flow Cytometry, Purification, Phospho-proteomics, Immunoprecipitation, Western Blot, Luciferase, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of CD112R as a novel checkpoint for human T cells

doi: 10.1084/jem.20150785

Figure Lengend Snippet: Expression of a putative ligand for CD112R. (A) Immune cells in human blood and monocyte-derived DCs were stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein by flow cytometry. Immune cells were gated on individual lineage markers. T cell: CD3; B cell: CD19; NK cell: CD56 + CD3 − ; monocyte: CD14; DC: CD11c. (B) Cultured human tumor cell lines were stained with control (FLAG-Fc) and CD112R protein, and the median fluorescence intensity (MFI) was determined by flow cytometry. CD112R MFI ratios (CD112R MFI/control MFI) are indicated. (C) SK-MEL28 melanoma line with or without trypsin treatment (10 min) was stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein by flow cytometry. (D) Blockade of CD112R binding by a CD112R mAb. HEK293T cells were stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein by flow cytometry with or without the presence of a CD112R mAb (clone 2H6). All data shown are representative of at least three independent experiments.

Article Snippet: Mouse CD112R cDNA (NCBI Reference Sequence accession no. XM_011240964 .1 ) was synthesized from GenScript and cloned onto a pcDNA3.1(−) expression vector.

Techniques: Expressing, Derivative Assay, Staining, Control, Flow Cytometry, Cell Culture, Fluorescence, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of CD112R as a novel checkpoint for human T cells

doi: 10.1084/jem.20150785

Figure Lengend Snippet: Identification of CD112 as a ligand for CD112R. (A) HEK293T cells were transiently transfected with different PVR-like gene plasmids as indicated and stained with control (FLAG-Fc; red) or CD112R-Fc (blue) protein. (B) HEK293T cells transduced with CD112R gene were incubated with anti-CD112R mAb (clone 2H6) or control mIgG1 as indicated before being stained with CD112-Fc (blue) or control FLAG-Fc (red). (C) Beads coated with CD112 (right) or control protein (left) were stained with CD112 mAb (blue) or isotype control (red) to confirm the presence of CD112 on beads. Beads were also incubated with CD112R fusion protein (blue) or control (red) for direct interaction. (D) Biacore 3000 analysis of CD112R binding to CD112. The surface plasmon resonance sensorgrams were recorded with threefold serial dilutions starting at the highest concentration of 333 nM. The fitting curves are in orange. (E) RMA-S/mCD112 (blue) or control RMA-S (red) cells were stained for binding by mCD112 mAb or mCD112R, mCD226, and mTIGIT fusion protein, respectively. (F) Competitive binding assay for CD112 among CD112R, CD226, and TIGIT proteins. Beads coated with CD112 were stained by CD112R-Fc protein in the presence of different concentrations of TIGIT or CD226 protein, whereas beads coated with CD112 were stained by CD226-Fc in the presence of different concentrations of CD112R protein. All data shown are representative of at least two independent experiments.

Article Snippet: Mouse CD112R cDNA (NCBI Reference Sequence accession no. XM_011240964 .1 ) was synthesized from GenScript and cloned onto a pcDNA3.1(−) expression vector.

Techniques: Transfection, Staining, Control, Transduction, Incubation, Binding Assay, SPR Assay, Concentration Assay, Competitive Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Identification of CD112R as a novel checkpoint for human T cells

doi: 10.1084/jem.20150785

Figure Lengend Snippet: CD112 binds to CD112R to inhibit T cell response. (A) Human monocyte–derived DCs stimulated with LPS overnight were preincubated with mIgG1 or CD112 mAb (clone TX31) and then stained for CD112R protein binding. DCs stained with control FLAG protein are shown in red. Data shown are representative of three different experiments ( n = 3 donors). (B) Human pancreatic cell line PANC198 was stained with isotype control (red) or CD112 mAb (blue) for CD112 expression (left). Cells were preincubated with control mIgG1 or CD112 mAb (clone TX31) before being stained by control (FLAG-Fc; red) or CD112R fusion protein (blue). Data shown are representative of two independent experiments. (C) Purified human T cells were CFSE labeled and stimulated with OKT3 together with plate-coated CD112-Fc or control protein (FLAG-Fc). Control (mouse IgG1) or CD112R mAb was added during cell culture. Cells were gated on CD8 + T cells, and their division was analyzed based on the dilution of CFSE. The CFSE-diluted cells indicated were counted as divided T cells. Data shown are representative of at least three independent experiments ( n > 3 donors). (D and E) CFSE-labeled CD4 + T cells were cultured with mytomycin-treated CHO stimulators expressing CD112 or control CHO stimulator cells. Antibodies as indicated were added from the beginning of culture. After 5 d of culture, cell division was analyzed based on the dilution of CFSE (D). (E) IL-2 (day 2) and other cytokines (day 5) in the supernatant were measured by a human T helper cytokine panel. Data are representative of three independent experiments. (F) Purified human T cells were labeled with CFSE and were co-cultured with autologous DCs in the presence of TT. Control (mouse IgG1), CD112R mAb, or TIGIT mAb was included at the beginning of the culture. The proliferation of TT-specific CD4 + T cells was determined by CFSE dilution of the human CD3 and CD4 double-positive cells. Data are representative of three independent experiments ( n = 3 donors). (G) In the same culture condition as in F, CD112R-Fc or control protein (FLAG-Fc) was included at the beginning to examine the effect on TT-specific T cell response. n = 5. *, P < 0.05 using two-way ANOVA. All bar graphs in C–F represent the mean ± SD results; *, P < 0.05; **, P < 0.01 (Student’s t test). The numbers in the histograms in C, D, F, and G refer to the percentages of divided T cells.

Article Snippet: Mouse CD112R cDNA (NCBI Reference Sequence accession no. XM_011240964 .1 ) was synthesized from GenScript and cloned onto a pcDNA3.1(−) expression vector.

Techniques: Derivative Assay, Staining, Protein Binding, Control, Expressing, Purification, Labeling, Cell Culture

Journal: Nature

Article Title: Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

doi: 10.1038/nature13441

Figure Lengend Snippet: Figure 2 | Mutant IDH blocks hepatocyte differentiation by silencing HNF- 4a. a, Heat map of GSEA showing top-ranked gene sets distinguishing IDH1(R132C) or IDH2(R172K) from wild-type (WT) or empty vector (EV) control hepatoblasts (pairwise analysis; replicates for each condition; see Methods). NES, normalized enrichment score; NS, not significant. b, c, Hepatoblasts analysed by immunoblot (b) and qRT–PCR (c). d, e, Analysis of wild-type hepatoblasts expressing the indicated short hairpin (sh)RNAs (uncoated plates). CTL, control. d, Hepatocyte sphere formation. e, Proliferation of shRNA-expressing hepatoblast cells co-expressing EV or shRNA-resistant Hnf4a(1) complementary DNA. f–h, Control and R132C- expressing hepatoblasts co-expressing vector control (EV2) or HNF-4a, grown on uncoated plates. f, Hepatocyte sphere formation. g, Hepatocyte gene expression. h, Proliferation. Scale bars, 100mm (d), 250mm (f). *P , 0.05.

Article Snippet: Human wild-type IDH1 cDNA (NCBI RefSeq accession number NM_ 005896.3) was obtained from Origene and subcloned into pRetro-Puro using EcoRI and BamHI fragments.

Techniques: Mutagenesis, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Expressing, shRNA, Gene Expression